The present invention relates generally to materials and methods for inhibition of Hodgkin and Reed Sternberg cell proliferation.
Hodgkin""s disease (HD) is unique among human lymphomas in that the tumor cells, known as Hodgkin and Reed Sternberg (H/RS) cells, are exceedingly rare, generally representing 0.1-1% of the total cell population within lymphoma tissue [Drexler, Leukemia and Lymphoma 8:283-313 (1992); Drexler, Leukemia and Lymphoma 9:1-25 (1993); Kadin, Current Opinion in Oncology 6:456:463 (1994); Cossman, et al., Lab. Invest 78:229-235 (1998)]. As a result, investigation of HRS cells has been impeded by their low frequency. PCR-based assays of DNA from single H/RS cells have revealed rearranged immunoglobulin genes and somatic mutations, suggesting that H/RS cells are clonal and may be derived from germinal center B cells [Kanzler, et al., J Exp Med 184:1495-505 (1996); Kyppers, et al., Annu Rev Immunol 16:471-93 (1998)]. The precise pathogenesis of HD, however, remains to be determined.
Previous observations suggest that the proliferation and survival of HD-derived cells depends on cytokine signaling. It is well established that the unique histology and eosinophilia of HD tissues, and secondary symptoms in the patient such as fever, weight loss, and night sweats, are induced by a pathological pattern of cytokine secretion [Drexler, Leukemia and Lymphoma. 8:283-313 (1992); Drexler, Leukemia and Lymphoma 9:1-25 (1993)]. For example, overexpression of IL-5 in H/RS cells has been previously demonstrated by in situ hybridization, but only in HD patients exhibiting eosinophilia [Samoszuk, et al., Blood 75:13-16 (1990); Samoszuk, Blood 79:1518-22 (1992)]. To date, no cytokine has been consistently reported as being overexpressed in HD-derived cell lines or in primary H/RS cells.
Evidence for a role for IL-13 in the etiology of HD is indirect. IgE is elevated in HD tissues and serum samples from HD patients [Samoszuk, Blood 79:1518-22 (1992); Thomas, et al., Ann Allergy 37:416-19 (1976)], and IL13 is known to promote Ig class switching to IgE. IL13-deficient mice exhibit lower basal levels of serum IgE [McKenzie, et al., Immunity 9:423432 (1998)]. Furthermore, studies of IL-4 deficient, IL-13 transgenic mice have demonstrated that IL-13 can promote class switching to IgE independently of IL-4[Emson, et al., J Exp. Med 188:399404 (1998)], emphasizing that IL-4 and IL-13 have distinct roles in regulating B cell functions.
IL-13 is a T cell-derived cytokine with immunomodulatory and anti-inflammatory properties [Minty, et al., Nature 362:248-250 (1993)]. The biological effects of IL-13 on B cells, macrophages, and monocytes are very similar to those of IL-4, probably because the IL-4 and IL-13 receptors share a common a chain. In B cells, IL-13 promotes proliferation, differentiation, and Ig heavy chain class switching to IgE and IgG4 [Zurawski, et al., Immunology Today 15:19-26 (1994)]. Proliferation results from a signaling pathway in which the engagement of the IL-13 receptor activates JAK1, which in turn activates STAT6 [Lin, et al., Immunity 2:331-339 (1995)].
Other aspects of the HD phenotype may also be attributable to the effects of IL-13. A recent study of IL-13-deficient mice has shown that cultures of type 2 helper T (Th2) cells from these animals produce significantly reduced levels of IL-4, IL-5, and IL-10 compared to the wild type, suggesting an important role for IL-13 as a regulator of Th2 cell commitment [McKenzie, et al, Immunity 9:423-432 (1998)]. If IL-13 is also important for promoting the differentiation of Th2 cells in humans, it could explain why H/RS cells (which secrete IL-13) are surrounded by Th2 cells in HD biopsies. In addition, because fibroblasts express the IL-13 receptor and can be activated by IL-13 [Doucet, et al., J Clin Invest 101:2129-2139 (1998)], the secretion of IL-13 by H/RS cells may underlie the pathogenesis of the fibrosis observed in nodular sclerosis HD.
Thus, there exists a need in the art to identify specific growth factors that participate in the pathogenesis of HD. In particular, identification of cytokines that stimulate H/RS cell proliferation will facilitate development of methods for therapeutic invention, by way of (i) modulating expression of the cytokine(s), (ii) modulating biological activity of the cytokine(s), and (iii) modulating both expression and biological activity.
The present invention provides materials and methods for inhibiting proliferation of cell types associated with Hodgkin""s disease. In particular, the invention contemplates inhibiting proliferation of Reed Sternberg cells found in individuals suffering from Hodgkin disease. Reed Sternberg cells associated with Hodgkin""s disease are referred to herein as H/RS cells.
The invention comprehends various methods by which proliferation of H/RS cells can be modulated, preferably through mechanisms that modulate expression, secretion, stimulation, activation, and/or biological activity of IL-13 and/or its cognate IL-13 receptor, as well as components in signal transduction pathways associated with IL-13 and the IL-13 receptor. In one aspect, the invention provides methods for inhibiting IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the step of contacting the H/RS cells with a compound that inhibits biological activity of IL-13. In a preferred embodiment, methods are provided wherein the compound that inhibits IL-13 biological activity is an antibody. It is preferred that the antibody is a monoclonal-antibody that specifically recognizes and binds to IL-13, and most preferably, the monoclonal antibody is a human antibody, however, humanized antibodies and non-human antibodies are also comprehended by the invention.
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The term xe2x80x9cspecifically recognizes or bindsxe2x80x9d as used herein indicates antibodies of the invention bind to only IL-3, IL-13 receptor, or naturally occurring variants thereof, despite any sequence similarity, homology, or identity conserved in other polypeptides.
In another aspect, the invention provides methods for inhibiting IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the step of contacting the H/RS cell types with a compound that inhibits biological activity of IL-13 receptors expressed on the H/RS cell types. In a preferred embodiment, methods are provided wherein the compound that inhibits IL-13 receptor biological activity is an antibody. It is preferred that the antibody is a monoclonal antibody that specifically recognizes and binds to the IL-13 receptor, and most preferably, the monoclonal antibody is a human antibody, however, humanized antibodies and non-human antibodies are also comprehended by the invention.
Alternatively, the invention provides methods for inhibiting IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the step of contacting the H/RS cells with a compound that inhibits expression of IL-13. In a preferred embodiment, methods of the invention are provided wherein the compound that inhibits IL-13 expression is an antisense polynucleotide that specifically recognizes a polynucleotide encoding IL-13. In another preferred embodiment, methods are provided wherein the compound that inhibits IL-13 expression is a ribozyme that binds to and acts specifically on polynucleotides encoding IL-13. The terms xe2x80x9cact specifically onxe2x80x9d and xe2x80x9cspecifically recognizes or bindsxe2x80x9d as used herein indicates that anti-sense and ribozymes of the invention bind to and act only on polynucleotides that encode IL-13, the IL-13 receptor, or naturally occurring variants thereof despite any sequence similarity, homology, or identity conserved in polynucleotides encoding other polypeptides.
Similarly, the invention also provides methods for inhibiting IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the step of contacting the H/RS cells with a compound that inhibits expression of IL-13 receptor. In a preferred embodiment, methods of the invention are provided wherein the compound that inhibits IL-13 receptor expression is an antisense polynucleotide that specifically recognizes a polynucleotide encoding the IL-13 receptor. In another preferred embodiment, methods are provided wherein the compound that inhibits IL-13 receptor expression is a ribozyme that binds to and acts specifically on polynucleotides encoding the IL-13 receptor.
The invention comprehends methods for identifying a compound that inhibits IL-13 dependent proliferation of Hodgkin and Reed Sternberg (HI/S) cell types comprising the steps of: a) measuring binding between IL-13 with IL-13 receptor in the presence and absence of a candidate inhibitor compound; and b) identifying the candidate compound as an inhibitor when binding between IL-13 and the IL-13 receptor is less in the presence of the candidate compound than in the absence of the candidate compound. The invention also provides methods for identifying a compound, or combination of compounds, that inhibits IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the steps of: a) contacting IL-13 with IL-13 receptor, b) measuring binding between IL-13 and the IL-13 receptor; c) repeating step (a) in the presence of a candidate inhibitor compound or compounds; d) repeating step (b) in the presence of the candidate compound or compounds; and e) identifying the candidate compound or compounds as an inhibitor when binding between IL-13 and the IL-13 receptor is less in the presence of the candidate compound than in the absence of the candidate compound. In a preferred embodiment of the method, IL-13 receptor is expressed on the cell surface of the H/RS cell types.
The invention also provide methods to identify a compound that inhibits IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the steps of: a) measuring IL-13 expression from growing H/RS cells in the presence and absence of a candidate inhibitor compound; and b) identifying the candidate compound as an inhibitor when lower levels of IL-13 expression are measured in the presence of the candidate compound than in an absence of the candidate compound. In one aspect, the invention provides methods to identify a compound or combination of compounds that inhibits IL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprising the steps of: a) growing H/RS cell types; b) measuring IL-13 expression; c) incubating the cell with a candidate inhibitor compound or compounds; d) measuring IL-13 expression with the candidate compound or compounds; and e) identifying the candidate compound or compounds as an inhibitor when lower levels of IL-13 expression are measured with the candidate compound or compounds than in an absence of the candidate compound or compounds. In a preferred embodiment of the method, the candidate inhibitor compound is an anti-sense polynucleotide that specifically binds to a polynucleotide encoding IL-13.
In methods of the invention in which IL-13 or IL-13 receptor biological activity is inhibited, numerous modes of action for a candidate Inhibitor are contemplated. In one aspect, inhibitors are provided that bind and neutralize IL-13. Preferably, an inhibitor acting by this mechanism prevents interaction between IL-13 and the IL-13 receptor. The invention comprehends mechanisms by which the inhibitor binding interaction induces a conformational change in IL-13, and binding that results in stearic hindrance preventing receptor recognition. The invention further comprehends inhibitors of the IL-13 receptor biological activity, which like IL-13 inhibitors, can function in a variety of ways. In one aspect, the IL-13 receptor inhibitor binds and neutralizes IL-13 activity. Preferably, the inhibitor would prevent interaction of the receptor with IL-13, or may prevent interaction of the IL-13 receptor with some downstream component of an IL-13 metabolic pathway that is required for, or participates in, H/RS cell proliferation. For example, it is known that IL-13 receptor activates JAK 1 and the invention therefore embraces inhibitors that prevent this activation, directly or indirectly.
The invention further embraces methods to inhibit activity of or interaction between, components in IL-13 pathways downstream from the IL-13 receptor that are required for, or at least participate in, H/RS cell proliferation.
In identification of inhibitors, the invention provides various sources for candidate compounds. There are a number of different libraries used for the identification of small molecule modulators, including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of structural analogs of known compounds or compounds that are identified as xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d via natural product screening. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant, or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opion. Biotechnol. 8:701-707(1997). Identification of modulators (xe2x80x9chitsxe2x80x9d) through use of the various libraries described herein permits modification of the candidate xe2x80x9chitxe2x80x9d (or xe2x80x9cleadxe2x80x9d) to optimize the capacity of the xe2x80x9chitxe2x80x9d to modulate activity. Methods of the invention are particularly amenable to high throughput screening (HTS) assays wherein large numbers of candidate inhibitors can been screen simultaneously, preferably by automated mechanisms.
In another aspect, the invention provides antibodies that inhibit IL-13 or IL-13 receptor activity. The invention provides monoclonal and polygonal antibodies, single chain antibodies, chimeric antibodies, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention. In a preferred embodiment, the invention provides human antibodies that specifically recognize IL-13, IL-13 receptor, other components in IL-13 pathways that participate in H/RS cell proliferation, and naturally occurring variants thereof. Human antibodies are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. The term xe2x80x9cspecifically recognizexe2x80x9d indicates that the variable regions of the antibodies recognize and bind, for example IL-13 or IL-13 receptor polypeptides, exclusively (i.e., able to distinguish IL-13 the IL-13 receptor, or naturally occurring variants thereof, from closely related polypeptides despite sequence identity, homology, or similarity found in the structure of the polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies that recognize and bind fragments of; for example, IL-13 or IL 13 receptor polypeptides of the invention, are also contemplated, provided that the antibodies are first and foremost specific for intact (ie., xe2x80x9cunfragmentedxe2x80x9d) polypeptides. As with antibodies that are specific for full length IL-13 or IL-13 receptor polypeptides (or other polypeptides of the invention), antibodies that recognize IL-13 or IL-13 receptor fragments are those which can distinguish IL-1-13 or IL-13 receptor polypeptides from other polypeptides despite inherent sequence and structural identity, homology, or similarity.
As still another aspect, the invention provides antisense polynucleotides which inhibit translation of a polynucleotide encoding IL-13, IL-13 receptor, other components of an IL-13 pathway that participate in H/RS cell proliferation, and naturally occurring variants thereof. Full length and fragment anti-sense polynucleotides are provided. The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to, for example either IL-13 or IL-13 receptor, RNA (as determined by sequence comparison of DNA encoding IL-13 or the IL-13 receptor to DNA encoding other known molecules and identification of characteristic or signature sequences) as well as (ii) those which recognize and hybridize to RNA encoding variants of the, for example, IL-13 or IL-13 receptor family of proteins. Antisense polynucleotides that hybridize to RNA encoding related members of the IL-13 or IL-13 receptor families of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for either family of molecules.
The invention further contemplates methods to modulate IL-13,IL-13 receptor, other components of an IL-13 pathway that participate in H/RS cell proliferation, and naturally occurring variants thereof, expression through use of ribozymes. For a review, see Gibson and Shillitoe, Mol. Biotech 7:125-137 (1997). Ribozyme technology can be utilized to inhibit translation of, for example, IL-13 or IL-13 receptor mRNA in a sequence specific manner through (i) the hybridization of a complementary RNA to a target mRNA and (ii) cleavage of the hybridized mRNA through nuclease activity inherent to the complementary strand. Ribozymes can be identified by empirical methods, but more preferably are specifically designed based on accessible sites on the target mRNA (Bramlage, et al., Trends in Biotech 16:434-438 (1998). Delivery of ribozymes to target cells can be accomplished using either exogenous or endogenous delivery techniques well known and routinely practiced in the art. Exogenous delivery methods can include use of targeting liposomes or direct local injection. Endogenous methods include use of viral vectors and non-viral plasmids.
Ribozymes can specifically modulate expression of IL-13, IL-13 receptor, other components of an IL-13 pathway that participate in H/RS cell proliferation, and naturally occurring variants thereof when designed to be complementary to regions unique to a polynucleotide encoding either polypeptide. xe2x80x9cSpecifically modulatexe2x80x9d therefore is intended to mean that ribozymes of the invention recognizes only a polynucleotide encoding, for example, either IL-13 or the IL-13 receptor. Similarly, ribozymes can be designed to modulate expression of all or some of the proteins closely related to either IL-13 or the IL-13 receptor. Ribozymes of this type are designed to recognize polynucleotide sequences conserved in all or some of the polynucleotides which encode the family of proteins.
The invention further embraces methods to modulate transcription of IL-13, IL-13 receptor, other components of an IL-13 pathway that participate in H/RS cell proliferation, and naturally occurring variants thereof, through use of oligonucleotide-directed triplet helix formation. For a review, see Lavrovsky, et al., Biochem. Mol. Med 62:11-22 (1997). Triplet helix formation is accomplished using sequence specific oligonucleotides which hybridize to double stranded DNA in the major groove as defined in the Watson-Crick model. Hybridization of a sequence specific oligonucleotide can thereafter modulate activity of DNA-binding proteins, including, for example, transcription factors and polymerases. Preferred target sequences for hybridization include promoter and enhancer regions to permit regulated transcription of IL-13, IL-13 receptor, other components of an IL-13 pathway that participate in H/RS cell proliferation, and naturally occurring variants thereof Oligonucleotides which are capable of triplet helix formation are also useful for site-specific covalent modification of target DNA sequences. Oligonucleotides useful for covalent modification are coupled to various DNA damaging agents as described in Lavrovsky, et al. [supra].
The invention further provides methods for treating Hodgkin""s Disease comprising the steps of administering to an individual in need thereof a therapeutically effective amount of a compound that inhibits IL-13 biological activity. Similarly, the invention contemplates methods for treating Hodgkin""s Disease comprising the steps of administering to an individual in need thereof a therapeutically effective amount of a compound that inhibits IL-13 expression. In a preferred method, the inhibitor is an antisense polynucleotide that specifically binds a polynucleotide encoding IL-13 or an antibody that specifically recognizes an IL-13 polypeptide.
The invention also comprehends methods for treating Hodgkin""s Disease comprising the step of administering to an individual in need thereof a therapeutically effective amount of a compound that inhibits IL-13 receptor biological activity. In another sect, the invention provides methods for treating Hodgkin""s Disease comprising the step of administering to an individual in need thereof a therapeutically effective amount of a compound that inhibits IL-13 receptor expression.
Inhibitors of the invention can be administered in combination with a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmacuetical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
The inhibitor compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the composition into the recipient organism and, particularly, when the composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
The inhibitor compositions may be introduced into the subject by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection; by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time. Optimal dosages may be readily determined by routine methods.
In another aspect, the invention contemplates use of an inhibitor of IL-13 expression in the production of a medicament for treatment of IL-13 dependent Hodgkin and Reed Sternberg cell proliferation. As another aspect, use of an inhibitor of IL-13 receptor biological activity in the production of a medicament for treatment of IL-13 dependent Hodgkin and Reed Sternberg cell proliferation is also provided. The invention also embraces use of an inhibitor of IL-13 receptor expression in the production of a medicament for treatment of IL-13 dependent Hodgkin and Reed Sternberg cell proliferation. In still another aspect, the invention comprehends use of an inhibitor of IL-13 biological activity in the production of a medicament for treatment of IL-13 dependent Hodgkin and Reed Sternberg cell proliferation.
While the invention is exemplified with respect to expression and biological activity of IL-13 and its cognate receptor, the invention also comprehends materials and methods to modulate expression and biological activity of other compounds (and their receptors where applicable) demonstrated herein to have high expression levels in H/RS cells, including for example, IL-5, ornithine decarboxylase (ODC), ICAM-3, urokinase (UPA), IgE Fc receptor II, insulin-like growth factor (IGF) II, NF-IL-3AE4BP4, Notch2, GM-CSF, interferon regulatory factors (IRF) 1, 5 and 6, nitric oxide synthase (NOS) 3, and the T cell receptor delta chain. The invention also comprehends materials and methods to modulate expression and biological activity of compounds in IL-13 pathways that participate, directly or indirectly, in H/RS cell proliferation.